Astrocytes
The combination of cell transplantation and gene transfer techniques provides a therapeutic approach to neurodegenerative diseases and traumatic injury. Several cell types, e.g. progenitor cells, neurons, glial cells, fibroblasts and myoblasts, have been investigated as vehicles for gene delivery to the central nervous system (CNS) (Wolff et al., 1989; Horellou et al., 1990a,b; Fisher et al., 1991, 1993, Gage et al., 1995; Sabate et al., 1995; Fisher, 1997; Martinez-Serrano and Bjorklund, 1997). Particular attention has been paid to the therapeutic activity of genetically engineered astrocytes (Cunningham et al. 1991, 1994; La Gamma et al., 1993; Castillo et al., 1994: Pundt et al., 1995; Lundberg et al., 1996; Lin et al., 1997). Astrocytes are especially targeted for brain repair because they are normal CNS constituents, are endowed with efficient secretory mechanisms and provide support to neurons through the release of trophic factors that promote their survival, differentiation and regeneration In addition, they can be expanded in culture and genetically engineered to express foreign transgenes. Recently, human fetal astrocytes arising from legal abortions have been cultured and successfully transduced with a retrovirus driving the expression of active NGF (Lin et al., 1997).
Human adult astrocytes are more relevant for human brain repair, since they allow autologous ex vivo gene transfer, thus obviating immunological rejection and side effects of immunosuppressors. In recent studies that attempted to culture human adult astrocytes, contamination with microglial cells was reported (Yong et al., 1991, 1992).
Gene Therapy
Ex vivo gene transfer involves the delivery of therapeutic cells to a patient (Wolff et al., 1989; Horellou et al., 1990a,b; Fisher et al., 1991, 1993, Gage et al., 1995; Sabate et al., 1995; Fisher, 1997; Martinez-Serrano and Bjorklund, 1997; Taylor, 1997). The use of immortalized cell lines for gene therapy is of limited value, since they retain tumorigenic properties. In contrast, primary cultured cells are more suitable. One of the best cell types for ex vivo gene transfer and subsequent transplantation is astrocytes (La Gamma et al., 1993; Castillo et al., 1994; Cunningham et al., 1994; Ridoux et al., 1994; Pundt et al., 1995; Lundberg et al., 1996; Lin et al., 1997), which are normally present in the CNS as the major supporting cells with efficient secretory machinery. Grafted .astrocytes, previously modified to produce enzymes, neurotransmitters or trophic factors, a can integrate and function suitably in the CNS (see refs in Taylor, 1997). Rat primary cultured astrocytes have been genetically engineered to produce NGF and BDNF, and survive after transplantation into the brain (Cunningham et al., 1991, 1994; Yoshimoto et al., 1995).
Recently, the interest in human astrocytes has grown (Yong et al., 1991, 1992: Aloisi et al., 1992; Perzelova and Mares, 1993; Pundt et al., 1995; Lin et al. 1997). Yong and colleagues (1992) reported that up to 80% of primary cultured cells were macrophages/microglial cells. After removal of most of the oligodendrocytes and microglial cells, 70% of the cells were astrocytes (Yong et al., 1992). The presence of microglial cells in astrocyte cultures may cause undesirable effects both in vitro and in vivo, since they proliferate actively and are major components of the immune cell population. Therefore, there is a need for a method to produce a population of astrocytes that is free of contaminating microglial cells. The present invention addresses this need, as discussed below. The present invention overcomes the disadvantages of impure preparations of astrocytes, and provides a cell preparation suitable for treating trama to the CNS and for the treatment of neurodegenerative disorders, such as Parkinson's, Huntington's or Alzheimer's disease.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.